Name: evaluating cell death using cell-free supernatant of probiotics in three-dimensional spheroid cultures of colorectal cancer cells
Uploaded: 2020-06-13T14:45:00
Description: Watch this Scientific Journal Video about Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells at JoVE.com

Denne forskningen ble støttet av "Etablering av målestandarder for kjemi og stråling", tilskuddsnummer KRISS-2020-GP2020-0003, og "Utvikling av målestandarder og teknologi for biomaterialer og medisinsk konvergens", tilskuddsnummer KRISS-2020-GP2020-0004 programmer, finansiert av Korea Research Institute of Standards and Science. Denne forskningen ble også støttet av Vitenskaps- og IKT-departementet (MSIT), National Research Foundation of Korea (NRF-2019M3A9F3065868), Helse- og velferdsdepartementet (MOHW), Korea Health Industry Development Institute (KHIDI, HI20C0558), Nærings- og energidepartementet (MOTIE) og Korea Evaluation Institute of Industrial Technology (KEIT, 20009350). ORCID ID (Hee Min Yoo: 0000-0002-5951-2137; Dukjin Kang: 0000-0002-5924-9674; Seil Kim: 0000-0003-3465-7118; Joo-Eun Lee: 0000-0002-2495-1439; Jina Lee: 0000-0002-3661-3701). Vi takker Chang Woo Park for hjelp med eksperimenter.

1. Bakterielle cellekulturer og tilberedning av Lactobacillus cellefri supernatant (LCFS)
MERK: Trinn 1.2 – 1.9 utføres i et anaerobt kammer.
<ol><li>Forbered en MRS agarplate og buljong som inneholder L-cystein og steriliser ved autoklavering.</li>
	<li>Pre-inkuber MRS agarplaten i H 2 anaerobtkammer opprettholdt ved 37 °C med 20 ppm oksygen.</li>
	<li>Tine Lactobacillus bakteriell bestand og inokuler agarplaten med bakteriekulturen (Fig...

<ol>
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Vevsmikromiljøet, inkludert nærliggende celler og den ekstracellulære matrisen (ECM), er grunnleggende for vevsgenerering og avgjørende for kontroll av cellevekst og vevsutvikling13. 2D-kulturer har imidlertid flere ulemper, for eksempel forstyrrelse av cellulære interaksjoner, samt endringer i cellemorfologi, ekstracellulære miljøer og tilnærmingen til divisjon14. 3D-cellekultursystemer har blitt grundig studert for bedre å reprodusere in vivo-effekter, og har bli...

Probiotika er de mest fordelaktige mikroorganismer i tarmen som forbedrer immun homeostase og vert energi metabolisme1. Probiotika fra Lactobacillus og Bifidobacterium er de mest avanserte av sitt slag som finnes i tarmen2,3. Tidligere undersøkelser har vist at Lactobacillus har hemmende og antiproliferative effekter på flere kreftformer, inkludert kolorektal kreft4. Videre forhindrer probiotika inflammatoriske tarmsykdommer, Crohns sykdom og ulcerøs kolitt5,6. Imidlertid ble de fleste studier med probiotika utført i todimensjonale (2D) monolayers som dyrkes på faste overflater.
Kunstige kultursystemer mangler miljøegenskaper, noe som ikke er naturlig for kreftceller. For å overvinne denne begrensningen er tredimensjonale (3D) kultursystemer utviklet7,8. Kreftceller i 3D viser forbedringer når det gjelder grunnleggende biologiske mekanismer, for eksempel celle levedyktighet, spredning, morfologi, cellecellekommunikasjon, legemiddelfølsomhet og in vivo relevans9,10. Videre er sfæroider laget av multicellulære typer kolorektal kreft og er avhengige av cellecelleinteraksjoner og den ekstracellulære matrisen (ECM)11. Vår forrige studie har rapportert at probiotisk cellefri supernatant (CFS) produsert ved hjelp av Lactobacillus fermentum viste anti-kreft effekter på 3D-kulturer av kolorektal kreft (CRC) celler12. Vi foreslo at CFS er en passende alternativ strategi for testing av probiotiske effekter på 3D-sfæroider12.
Her presenterer vi en tilnærming som kan imøtekomme multicellulære typer 3D-kolorektal kreft for analyse av terapeutiske effekter av probiotisk cellefri supernatant (CFS) på flere 3D-kolorektale kreftimileringssystemer. Denne metoden gir et middel for analyse av relaterte probiotiske og anti-kreft effekter in vitro.

<table>
	<tbody>
		<tr>
			<td> 
			10% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 &#181;l</td>
			<td>Biorad</td>
			<td>4561036</td>
			<td>Pkg of 10</td>
		</tr>
		<tr>
			<td>Applied Biosystems MicroAmp Optical Adhesive Film</td>
			<td>Thermo Fisher Scientific</td>
			<td>4311971</td>
			<td>100 covers</td>
		</tr>
		<tr>
			<td>10x transfer buffer</td>
			<td>Intron</td>
			<td>IBS-BT031A</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>10X Tris-Glycine (W/SDS)</td>
			<td>Intron</td>
			<td>IBS-BT014</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Axygen 2.0 mL MaxyClear Snaplock Microcentrifuge Tube, Polypropylene, Clear, Nonsterile, 500 Tubes/Pack, 10 Packs/Case</td>
			<td>Corning</td>
			<td>SCT-200-C</td>
			<td>500 Tubes/Pack, 10 Packs/Case</td>
		</tr>
		<tr>
			<td>BD Difco Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>BD Difco Lactobacilli MRS Broth</td>
			<td>BD</td>
			<td>DF0881-17-5</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D Cell viability assay</td>
			<td>Promega</td>
			<td>G9681</td>
			<td>100&#956;l/assay in 96-well plates</td>
		</tr>
		<tr>
			<td>Complete Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11697498001</td>
			<td>vial of 20 tablets</td>
		</tr>
		<tr>
			<td>Corning Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 &#177; 0.1</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>EMD Millipore Immobilon-P PVDF Transfer Membranes</td>
			<td>fisher Scientific</td>
			<td>IPVH00010</td>
			<td>26.5cm x 3.75m roll; Pore Size: 0.45um</td>
		</tr>
		<tr>
			<td>Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>25/Pack, 500/Case</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, US origin</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000044</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit, 25 x 20 &#181;l rxns #1708890</td>
			<td>Biorad</td>
			<td>1708890</td>
			<td>25 x 20 &#181;L rxns</td>
		</tr>
		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>Biorad</td>
			<td>1725121</td>
			<td>5 x 1 mL</td>
		</tr>
		<tr>
			<td>Lactobacillus fermentum</td>
			<td>Korean Collection for Type Cultures</td>
			<td>KCTC 3112</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine hydrochloride monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C6852-25G</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>Methyl Cellulose (3500-5600mPa&#183;s, 2% in Water at 20&#176;C)</td>
			<td>TCI</td>
			<td>M0185</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL</td>
			<td>Applied Biosystems</td>
			<td>4346906</td>
			<td>20 plates</td>
		</tr>
		<tr>
			<td>Millex-GS Syringe Filter Unit, 0.22 &#181;m, mixed cellulose esters, 33 mm, ethylene oxide sterilized</td>
			<td>Millipore</td>
			<td>SLGS033SB</td>
			<td>250</td>
		</tr>
		<tr>
			<td>PE Annexin V Apoptosis Detection Kit with 7-AAD</td>
			<td>Biolegend</td>
			<td>640934</td>
			<td>100 tests</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Introgen</td>
			<td>P1304MP</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>89901</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit (250)</td>
			<td>Qiagen</td>
			<td>74106</td>
			<td>250</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200056</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Name of Materials/Equipment/Software</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>anti - p-I&#954;B&#945; (B-9)</td>
			<td>Santa cruze</td>
			<td>sc-8404</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>anti-BclxL (H-5)</td>
			<td>Santa cruze</td>
			<td>sc-8392</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>anti-PARP 1 (C2-10)</td>
			<td>Santa cruze</td>
			<td>sc-53643</td>
			<td>50 &#181;l ascites</td>
		</tr>
		<tr>
			<td>anti-&#946;-actin (C4)</td>
			<td>Santa cruze</td>
			<td>sc-47778</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>BD FACSVerse</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>San Diego, CA, USA</td>
		</tr>
		<tr>
			<td>Synergy HTX Multi-Mode Microplate Reader</td>
			<td>BioT</td>
			<td>S1LFA</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo fisher</td>
			<td>HERAcell 150i</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube 15 ml</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube 50 ml</td>
			<td>SPL</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Ultra-Low Attachment Multiple Well Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS7007</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Ultra-Low Attachment Multiple Well Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 50 mL Reagent Reservoirs, 5/Bag, Sterile</td>
			<td>Costar</td>
			<td>4870</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Thermofisher</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>invitrogen</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>EnSpire Multimode Reader</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Enspire 2300</td>
		</tr>
		<tr>
			<td>Eppendorf Research Plus Multi Channel Pipette, 8-channel</td>
			<td>Eppendorf</td>
			<td>3122000051</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo software</td>
			<td>TreeStar</td>
			<td></td>
			<td>Ashland, OR, USA</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson immunoresearch</td>
			<td>115-035-062</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG (H+L)</td>
			<td>Jackson immunoresearch</td>
			<td>111-035-144</td>
			<td>2.0 mL</td>
		</tr>
		<tr>
			<td>GraphPad Prism 5</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>Inc., San Diego, CA, USA</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageQuant LAS 4000 mini</td>
			<td>Fujifilm</td>
			<td></td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Incubated shaker</td>
			<td>Lab companion</td>
			<td>SIF-6000R</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi Gauge Ver. 3.0,</td>
			<td>Fujifilm</td>
			<td></td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Optical density (OD)LAMBDA UV/Vis Spectrophotometers</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Waltham, MA, USA</td>
		</tr>
		<tr>
			<td>Phase-contrast microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>SPL microcentrifuge tube 1.5mL</td>
			<td>SPL</td>
			<td>60015</td>
			<td></td>
		</tr>
		<tr>
			<td>SPL Multi Channel Reservoirs, 12-Chs, PS, Sterile</td>
			<td>SPL</td>
			<td>21012</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOnePlus Real-Time PCR system</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Waltham, MA, USA</td>
		</tr>
		<tr>
			<td>Vibra-Cell Ultrasonic Liquid Processors</td>
			<td>SONICS-vibra cell</td>
			<td>VC 505</td>
			<td>500 Watt ultrasonic processor</td>
		</tr>
		<tr>
			<td>Vinyl Anaerobic Chamber</td>
			<td>COY LAB PRODUCTS</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating cell death using cell-free supernatant of probiotics in three-dimensional spheroid cultures of colorectal cancer cells

Dette manuskriptet beskriver en protokoll for å evaluere kreftcelledødsfall i tredimensjonale (3D) sfæroider av multicellulære typer kreftceller ved hjelp av supernatanter fra Lactobacillus fermentumcellekultur , betraktet som probiotiske kulturer. Bruk av 3D-kulturer for å teste Lactobacillus cellefrie supernatant (LCFS) er et bedre alternativ enn testing i 2D-monolayers, spesielt ettersom L. fermentum kan produsere antikrefteffekter i tarmen. L. fermentum supernatant ble identifisert å ha økte anti-proliferative effekter mot flere kolorektal kreft (CRC) celler i 3D kulturforhold. Interessant nok var disse effektene sterkt relatert til kulturmodellen, og demonstrerte L. fermentums bemerkelsesverdige evne til å indusere kreftcelledød. Stabile sfæroider ble generert fra forskjellige CRCer (kolorektal kreftceller) ved hjelp av protokollen presentert nedenfor. Denne protokollen for å generere 3D-sfæroid er tidsbesparende og kostnadseffektiv. Dette systemet ble utviklet for å enkelt undersøke anti-kreft effekter av LCFS i flere typer CRC sfæroider. Som forventet, CRC sfæroider behandlet med LCFS sterkt indusert celledød under eksperimentet og uttrykte spesifikke apoptose molekylære markører som analysert av qRT-PCR, western blotting, og FACS analyse. Derfor er denne metoden verdifull for å utforske celle levedyktighet og evaluere effekten av anti-kreftmedisiner.

Vi beskriver protokollen for å skaffe sfæroider fra forskjellige kolorektal kreftcellelinjer. Tilskudd med metylcellulose var nødvendig for å generere sfæroider. Vi presenterer også en metode for LCFS-forberedelse og presenterer en modell for å studere sammenhengen mellom probiotika og kolorektal kreft. Sfæroiddannelse og LCFS-forberedelsesprotokoller er skjematisk illustrert i figur 1A,B. Som vist i figur 2Aforvandler metylcellulosekons...

Watch this Scientific Journal Video about Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells at JoVE.com

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

Her presenteres metoder for å forstå antikrefteffekter av Lactobacillus cellefri supernatant (LCFS). Kolorektal kreftcellelinjer viser celledødsfall når de behandles med LCFS i 3D-kulturer. Prosessen med å generere sfæroider kan optimaliseres avhengig av stillaset, og analysemetodene som presenteres er nyttige for å evaluere de involverte signalveiene.

Evaluering av celledød ved hjelp av cellefri supernatant av probiotika i tredimensjonale sfæroidkulturer av kolorektale kreftceller

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Research

JoVE Journal

Cancer Research

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

Utnytte Funksjonell genom Screening for å identifisere potensielt Novel narkotika mål i Cancer Cell spheroid kulturer

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Forberedelse og metabolske analysen av 3-dimensjonale Spheroid co kulturer av kreft i bukspyttkjertelen celler og fibroblaster

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Flytte oppover: En enkel og fleksibel In Vitro tredimensjonale invasjonen analysen protokoll

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Fullt Human tumor-baserte Matrix i tredimensjonale spheroid Invasion analysen

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Vurdering Cell levedyktighet og død i 3D spheroid kulturer av kreftceller

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

Vurderer differensiering kapasitet på Mouse prostata epitelceller bruke Organoid kultur

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Isolering av Stem-lignende celler fra 3-dimensjonale spheroid kulturer

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Påvisning av cellefritt DNA i blodplasmaprøver av kreftpasienter

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

En tredimensjonal modell av sfæroider for å studere kolonkreft stamceller

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Tredimensjonal in vitro biomimetisk modell av neuroblastom ved bruk av kollagenbaserte stillas

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...